1. Field of the Invention
This invention resides in the field of gel electrophoresis, and relates in particular to the transfer of electrophoretically separated species from a slab gel in which the species were separated to a sheet-form support matrix in which the species can be detected, identified, and in some cases, quantified.
2. Description of the Prior Art
The electroblotting of proteins, nucleic acids, or biological species in general from an electrophoresis slab gel to a membrane of nitrocellulose, nylon, polyvinyl difluoride, or similar materials is a common means by which a biochemist can identify the components of, and otherwise characterize, essentially any biological mixture. In electroblotting, the flat surfaces of the gel and membrane are placed in full direct contact and an electric current is passed through them in a direction transverse to the gel and membrane to transfer the species in the same way that the species were mobilized within the gel when an electric current was passed in a direction parallel to the gel. When the species are DNA fragments, the transfer is termed a Southern blot after its originator, the British biologist Edwin M. Southern. By analogy, the electroblotting of RNA fragments is termed Northern blotting, and the electroblotting of proteins or polypeptides is termed Western blotting. The analytical procedures that are performed on the species in the membrane are those that are appropriate to the type of species that are transferred. For example, in Southern and Northern blotting analysis is begun by the treatment of the species on the transfer membrane with a hybridization probe followed by labeling with a fluorescent or chromogenic dye, while in Western blotting, the analysis begins with treatment of the target proteins with antibodies.
Electroblotting of either the Southern, Northern, or Western type requires electrodes on either side of the gel and membrane stack and a source of ions to carry the electric current, and is typically performed in either a wet or a semi-dry format. In wet blotting, buffer solutions placed between the electrode and the gel or membrane serve as the source of ions, while in semi-dry blotting, the buffer solutions are replaced with filter papers wetted with the buffer solution. Semi-dry blotting therefore uses a transfer stack, also referred to as a “blotting sandwich,” that consists of, in order, a first sheet of buffer-wetted filter paper, the blotting membrane, the gel, and a second sheet of buffer-wetted filter paper. A “dry” electroblotting system is disclosed in an International Application published under the Patent Cooperation Treaty, Publication No. WO 2006/091525 A2, international publication date 31 Aug. 2006, entitled “Electro-blotting Devices, Systems, and Kits, and Methods for Their Use,” Invitrogen Corporation, applicant. uses no liquid buffers other than those residing in the gels, The ion reservoirs in this “dry” system are anodic and cathodic gel matrices that are placed between the corresponding electrode and the transfer stack, which consists only of the electrophoresis gel and the membrane. The system is termed “dry because it uses no liquid buffers other than those residing in the gel matrices that serve as the ion reservoirs.
One of the hazards encountered in electroblotting is heat generation due to the electric current. Excessive heat generation can cause damage to the proteins or other species being transferred, or to the gel, and nonuniform heating can result in unreliable or nonuniform transfers. Heat generation can be limited by using a low current or a buffer of low ionic strength, but these can result in incomplete transfers or in an excessive amount of time for completion of the transfer.